Separating method and an apparatus performing such a method

ABSTRACT

The present invention relates to a method for separating carriers from a solution, said carriers being adapted to have biomolecules, such as DNA, RNA, proteins, polypeptides or carbohydrates attached thereto. The method comprises the steps of introducing a tubular member ( 13 ) into a receptacle which holds a solution containing said carriers immersing an end portion of the tubular member in the solution attracting and holding said carriers to said end portion and removing said tubular member together with the held carriers from the solution. The method is characterised in that said attracting and holding step comprises the step of providing an under-pressure within the tubular member so as to attract and hold the carriers to a filter ( 25 ) being disposed in the end portion of the tubular member. The invention also relates to an apparatus ( 10 ) performing such a method.

FIELD OF THE INVENTION

The present invention generally relates to a method for separatingcarriers from a solution; said carriers being adapted to havebiomolecules attached thereto, and more particularly to a method usingsuch a separation for the preparation of single stranded samples ofgenetic material. The present invention also relates to an apparatus forperforming such methods. Finally, the present invention also relates toa disposable unit adapted to be attached to such an apparatus.

BACKGROUND OF THE INVENTION

Methods for analysing genetic material often require amplification ofthe nucleic acid sample as an initial step. This sometimes require thestep of separating the two strands of a double-stranded DNA sample(dsDNA). In doing so, a related art method known as the Polymerase ChainReaction (PCR) is employed. According to one variant of this method thedsDNA sample is amplified by the supply of a primer pair of which one isbiotinylated, to a receptacle, which holds the solution containing thedsDNA sample. Streptavidin coated beads are then added to the solution,and by also adding a binding buffer the amplified dsDNA will beimmobilised on the beads.

These beads may be made of sepharose, and one related art method forpreparing single stranded DNA (ssDNA) is as follows: First, thereceptacle holding the immobilised dsDNA is put on a vacuum station. Thebottom of the receptacle is provided with a filter, which pore diameteris less than the size of the sepharose beads. Thus, when anunder-pressure is applied on the underside of the filter the beads inthe solution will be left on the filter, while the solution is drainedaway through the filter to, e.g. a liquid collector. In a next step,sodium hydroxide is supplied to the receptacle, which will separate thestrands of the dsDNA into ssDNA, whereupon the sodium hydroxide isdrained away. Thus, the ssDNA strands which are not bound to the beadsare also drained away, while the bound ssDNA strands remain caught onthe filter. In yet a further step, remaining sodium hydroxide isneutralised by means of an addition of washing buffer one or severaltimes, and in a final step, in which the under-pressure is removed, asolution for re-suspending the caught beads in the solution is added.These beads having attached ssDNA strands are now ready to betransferred from this receptacle to an other receptacle for furtherpreparation, or for analysis of the ssDNA.

However, it is difficult to fully re-suspend all beads in the solutionsince the beads are caught in the filter, and easily stick there despitethe re-suspension attempts. Thus, trying to catch all beads by means ofpipetting is difficult. Moreover, the situation is made worse when theheights of the liquid columns of the wells are low which yet renderdifficulties to the pipetting. Furthermore, the receptacle is commonlyin the form of a multi-well receptacle, such as a Micro Titre Plate(MTP), having e.g. 96, 384, 1536 or 6144 separate wells, whereby arepetitive pipetting is disadvantageous from a strain injury point ofview.

An other related art method disclosed in GB 99 233 249 uses magneticbeads instead of sepharose beads. According to this method a magnet isintroduced into the receptacle, e.g. a well of a MTP, whereby thestreptavidin coated magnetic beads are attracted to and hold by themagnet. Thus, the magnetic beads as well as the bound dsDNA may beremoved from this receptacle and transferred to an other receptacle, inwhich e.g. a strand separating solution, such as sodium hydroxide iscontained. Accordingly, the dsDNA will be separated into ssDNA, wherebythe first strands are suspended in the solution, and optionally drainedaway, while the second strands remain bound to the beads. These latterstrands may now be transferred to an other receptacle for furtherpreparation, or for analysis.

However, magnetic beads are more expensive than sepharose beads, and theuse of sepharose beads also results in better quality in asequencing-by-synthesis method.

Furthermore, another related art method disclosed in U.S. Pat. No.6,156,550 is related to transferring beads from one solution to anothersolution. This method describes how sepharose beads can be attached to asurface of a polymer. The drawback with beads bound to a support is thatbeads bound to a surface do not behave biochemically or physicallysimilar to beads in a solution, thus further analysis of the ssDNA willbe difficult.

In an other related art method disclosed in JP 58223759 beads are movedbetween two test tubes. A nozzle 16 is immersed in a first test tube 17where it attracts a bead B by means of a vacuum. Then, the nozzle 16 ismoved to a second test tube 18 where a back pressure is applied so as torelease the bead B in the second test tube. During the whole movementthe vacuum prevails within the nozzle. However, the nozzle is onlycapable of moving large beads, which are attracted to and stuck on thetip of the nozzle. In case of small beads, such beads would be drainedaway together with the liquid contained in the test tubes.

OBJECT OF THE INVENTION

An object of the present invention is to provide an improved method forseparating carriers such as sepharose beads from solutions, said beadsfunctioning as carriers for biomolecules.

SUMMARY OF THE INVENTION

This object is achieved according to the present invention by means of amethod for separating carriers from a solution, said carriers beingadapted to have biomolecules, such as DNA, RNA, proteins, polypeptidesor carbohydrates attached thereto, wherein the method comprises thesteps of introducing a tubular member into a receptacle which holds asolution containing said carriers; immersing an end portion of thetubular member in the solution; attracting and holding said carriers tosaid end portion; removing said tubular member together with the heldcarriers from the solution. The method is characterised in that saidattracting and holding step comprises the step of providing anunder-pressure within the tubular member so as to attract and hold thecarriers to a filter being disposed in the end portion of the tubularmember.

This object is also achieved by means of an apparatus for movingcarriers between different receptacles, said carriers being adapted tohave biomolecules, such as DNA, RNA, proteins, polypeptides orcarbohydrates attached thereto, wherein the apparatus comprises at leastone tubular member to be introduced into a receptacle, which contains asolution containing said carriers, said tubular member having an endportion to be immersed in the solution; and means for attracting andholding said carriers to said end portion. The apparatus ischaracterised in that said means comprises a filter being disposed inthe end portion.

This object is also achieved by a method for preparing single strandedDNA (ssDNA) from double stranded DNA (dsDNA) comprising the steps ofimmobilising one of two strands of said dsDNA on carriers in a firstreceptacle holding a first solution, wherein the solution contains saidcarriers and said dsDNA; attracting and holding said carriers to atubular member being immersed in the first solution; moving said tubularmember together with the held carriers from the first receptacle to asecond receptacle; separating the dsDNA into ssDNA in the secondreceptacle; removing the tubular member from the second receptacle, thusremoving carriers with immobilised ssDNA from the second receptacle. Themethod is characterised in that said attracting and holding stepcomprises the step of providing an under-pressure within the tubularmember so as to attract and hold the carriers to a filter being disposedin the tubular member.

This object is also achieved by a disposable unit adapted to be attachedto the tubular member of an apparatus according to the preamble of claim9, wherein said disposable unit is provided with a filter for holdingsaid carriers.

By such a method and apparatus, carriers such as streptavidin coatedsepharose beads may be transferred between different receptacles holdingdifferent solutions. Hereby, the whole procedure of preparing e.g. ssDNAor other manual sample preparation is simplified as well as lesslabour-intensive in comparison with the related art sepharose method,but also less expensive since the use of magnetic beads is avoided. Ofcourse it is still possible to use the inventive methods and apparatustogether with magnetic beads functioning as carriers for biomolecules.

Suitably, said methods comprise the step of releasing said carriers fromthe filter by removing the under-pressure or creating an over-pressurewithin the tubular member. Hereby, release of the carriers from thefilter is enhanced.

Advantageously, each of the aforementioned steps is performedsimultaneously at a plurality of locations. Hereby, the methods areadapted for use with a multi-well receptacles, such as Micro TitrePlates (MTR) with 96, 384, 1536 or 6144 wells.

Preferably, the method is used for purifying proteins, and morepreferably for purifying tagged proteins, and most preferably forpurifying His-tagged proteins. Hereby is achieved that a multi-wellformat purification could be applied for e.g. screening of recombinantbacteria for protein production.

Suitably, said apparatus comprises a housing having a sealed inner spacewhich is connectable to a vacuum supply for creating an under-pressureinside the inner space, wherein the tubular member is in fluidcommunication with said inner space, and more suitably the tubularmember projects into the inner space. Due to the projections, thesolution will not flow back if the under-pressure is removed from theinner space, which prevent mixing of different solutions.

Preferably, the apparatus comprises a plurality of tubular membershaving a mutual arrangement corresponding to the arrangement of amulti-well receptacle. Hereby, the apparatus may be used with MicroTitre Plates (MTP).

Advantageously, all the tubular members are integrated in a separatesupport unit, which is detachable from the housing. Hereby, a disposableunit is provided. Furthermore, cleaning of tubes and filters issimplified.

Suitably, the apparatus comprises means or creating an over-pressurewithin the inner space. Hereby, the release of carriers which aretrapped in the filter is facilitated.

Preferably, the apparatus comprises means for controlling theunder-pressure and/or the over-pressure. Hereby, adaptation to differentappliances is achieved.

Advantageously, the methods and/or the apparatus are used in anautomatic robot system. Hereby, performance is improved since littlehuman intervention is required. Accordingly, a robotic arm may carry theapparatus so as to, in an automated and non-human controlled way,transfer carriers between solutions and/or wells. In this respect, thereleasing step may also be performed by shaking, vibrating or scrapingso as to release the carriers from the filter after the under-pressurehas been removed.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will now be describedwith reference to the accompanying drawings, on which:

FIG. 1 a shows a perspective view of an apparatus according to apreferred embodiment of the invention;

FIG. 1 b shows an exploded view of the apparatus in FIG. 1 a;

FIG. 2 a shows a top view of the apparatus;

FIG. 2 b shows a top view of the apparatus with a top cover beingremoved;

FIG. 2 c shows a view along section B-B of FIG. 2 a;

FIG. 2 d shows a view along section C-C of FIG. 2 a;

FIG. 3 shows a front view of a disposable unit according to anotheraspect of the present invention, and

FIGS. 4 a-b show SDS-PAGE analysis of eluted proteins, where FIG. 4 ashows CBB stain and FIG. 4 b shows Silver stain.

DETAILED DESCRIPTION

FIGS. 1 a-b show perspective views of an apparatus 10 in accordance witha preferred embodiment of the invention. The apparatus comprises a topcover 1 and a bottom cover 3 which together form a housing 5. A seal 7is arranged between the top and bottom covers. A conduit 9 is in one endconnected to the housing via a coupling 11, and in an other endconnectable to, e.g. a not shown vacuum supply, a liquid separatingdevice and/or a pressure supply. A plurality of tubular elongatedmembers in the form of tubes 13 are each connected to openings 15arranged on an underside 17 of the bottom cover 3. Sealing rings 19 areprovided in each opening for sealing against the tubes 13. The tubes arearranged in parallel rows and columns corresponding to the arrangementof the wells of a multi-well receptacle, such as a Micro Titre Plate(MTP). A support plate 21 is by means of a screw 22 attached to theunderside of the bottom cover, wherein the support plate is providedwith a plurality of holes 23 adapted for the tubes and aligned with theopenings in the bottom cover. A filter 25 is disposed in a cross-sectionin an end portion of each tube 13.

As can be seen from FIG. 2 c an inner space 27 is created between thetop 1 and bottom 3 covers when the top and bottom covers are assembled.This inner space is in fluid communication with the conduit 9 via arecess 29 arranged in a wall portion of the housing, and accordinglyalso in fluid communication with e.g. the vacuum supply mentionedearlier. The openings 15 of the bottom cover extend into the innerspace, whereby each tube is in fluid communication with the inner space(this is shown in FIG. 2 c for one or the tubes). As can be seen fromFIGS. 2 b-c, each tube also projects a short distance into the innerspace, thus forming chimney-like protrusions 31.

Operation

The operation of the apparatus will be described solely by means of anexample explaining the preparation of single stranded DNA samplestypically comprising 50-2000 nucleotides. The skilled person willhowever realise that the apparatus is not restricted to this applianceonly, but can be used in a number of appliances where carriers ofbiomolecules need to be separated from a solution or moved betweendifferent receptacles. As an example, even though streptavidin-biotinhave been employed as binding molecule pair, in the above mentionedexample, other binding molecules are also conceivable.

In the example, a multi-well receptacle, such as a MTP is provided (notshown), where each well holds a solution containing amplifieddouble-stranded DNA samples, which have been immobilised on beads, suchas streptavidin coated sepharose beads. The apparatus 10 is positionedabove the MTP and subsequently lowered, whereby the tubes 13 of theapparatus are inserted into the wells of the MTP and then immersed inthe solution. The vacuum supply is actuated whereby an under-pressure isprovided in the inner space 27 of the housing 5, and thus also in eachtube on a first side of the filter 25. By choosing a suitableunder-pressure the solution is drawn into the tubes and through thefilters. The solution enters the inner space and is subsequently drainedaway through the conduit 9 to a liquid separating device. The carrierswith the immobilised beads are also drawn towards the filter, but arecaught on a second side, i.e. opposite the first side, of the filter,while the solution is drained away through the filter. The filter ispreferably made of a sintered material, such as sintered polypropyleneplastic even though other materials are conceivable, and when the beadsare made of sepharose having a diameter of approximately 38 μm thefilter pore diameter needs to be about 10 μm.

The apparatus 10 may now be raised, with maintained under-pressure, thusremoving the tubes having carriers held to the filter. The apparatus ismoved to an other receptacle holding yet a solution in which e.g. astrand separating solution, such as sodium hydroxide is contained.Accordingly, the dsDNA will be separated into ssDNA when the tubes areimmersed in the solution, whereby the first strands are suspended in thesolution, while the second strands remain bound to the beads. Theapparatus is then moved to an other MTP, wherein each well holds yet asolution. The tubes are immersed into the solution of the wells, and thevacuum supply is inactivated whereby the carriers are released from thefilter and are re-suspended in the solution. The chimney-likeprotrusions 31 serve as to prevent solution contained in the inner spacefrom flowing back to a receptacle when the vacuum supply is inactivated.Such back flow would otherwise risk to contaminate the solution presentin a subsequent receptacle.

To facilitate the suspension the filter may be scraped against thebottom of the wells. The apparatus may also be shook or vibrated tofurther enhance the release of carriers. Alternatively, a pressuresupply may be actuated, whereby an over-pressure is provided in theinner space of the housing, and thus also on the first side of eachfilter. This reversion of the pressure will further enhance the releaseof carriers from the second side of the filter by a blowing actionthrough the filter, but also by creating a turbulent motion in thesolution, thus having a rinsing effect of the filter. When the carriersare suspended in the solution, the pressure supply is inactivated, andthe apparatus is raised thus removing the tubes from the wells. Now, thessDNA preparation is completed, and suitable analysis of the ssDNAcontained in the solution may commence. Such analysis may be sequencingor sequencing-by-synthesis as described in U.S. Pat. No. 5,405,746 andWO 98/13523, respectively.

After use of the apparatus cleaning of the filters may be required. Thetubes are simply immersed in e.g. water or ethanol so as to rinse thefilters, even though the. apparatus may be disassembled to completelyclean the apparatus. To further improve the cleaning effect, the vacuumsupply and/or the pressure supply may be actuated. In this case analternation between an under-pressure and an over-pressure may beperformed, which would improve the cleaning effect.

It is of course conceivable to design the support plate with integratedtubes and filters so as to create a disposable unit, which is thrownaway after use. Such an unit is easily exchanged if required.

It is also conceivable to displace the filters a short distance from theoutermost end portion of each tube. Thus, the capacity of holdingcarriers may be increased in case of solutions haling a high carrierdensity, or if high liquid columns occur in each well.

It is also possible that the carriers can be removed from the filter inthe tubular member by treating them with ultrasound, for example byplacing them in an ultrasonic water bath. In this way, the tubularmembers and filters will be cleaned before next usage.

Moreover, it is also conceivable to provide the tubular members 13 withdisposable tip units 50. Such an unit is shown in FIG. 3 and has a firstend 52 to be connected to the tubular member 13 and a second end 54 tobe immersed in the solution of a receptacle. The unit 50 has a taperedshape and is provided with a filter 25 for holding the carriers asdescribed above, thus it is not necessary to provide the tubular membersthem selves with filters. These units are disposable and normallyrejected after use.

Experiment

A study was also performed where the purpose was to investigate if itwas possible to purify a His-tagged protein (HisKlenow) from a bacteriallysate with good selectivity and yield. In the description below, theuse of the apparatus according to the present invention will only bedescribed. in a general way and is only referred to as the inventiveapparatus. The skilled man will however realise in conjunction with theDETAILED DESCRIPTION and OPERATION sections above how individual stepsduring purification of His-tagged proteins are to be performed with theaid of the inventive apparatus.

His-Klenow is the Klenow fragment of DNA polymerase with hexa-Histidineadded N-terminally. The protein is over expressed in E. coli forproduction purposes. The introduced modification makes it possible topurify the protein by immobilized metal affinity chromatography (IMAC),using commercially available matrices. The procedure involves binding ofthe protein to immobilized Ni²⁺-ions, washing out unbound substances andfinally elution of the bound protein with buffer containing 0.2 Mimidazol.

The study was performed by adoption of an related art method forchromatographic purification of His-Klenow to the inventive apparatusformat. Since all critical parameters such as binding and elutionproperties was established, it was not necessary to study these further.The only variable was the amount of gel added to each well since thiscould be suspected to influence recovery. Materials HiTrap Chelating (1ml) column Amersham Biosciences Lysate His-Klenow batch 1012 PCR-plates,96-welll Millipore NaH₂PO₄ Merck NaCl Merck Imidazol USB The inventiveapparatus Pyrosequencing Phast electrophoresis equipment AmershamBiosciences Phast 10-15% gels Amersham Biosciences SDS buffer stripsAmersham Biosciences Low Molecular Weight Amersham Biosciencescalibration kit Commassie Brilliant Blue stain Amersham BiosciencesPhast Silver stain kit Amersham Biosciences EDTA Merck Deionized waterNiCl₂x6(H₂O) SigmaBuffers

Binding and wash buffer: 10 mM NaP_(i), 0.5 M NaCl, 10 mM imidazol, pH7.4

Elution buffer: 10 mM NaP_(i), 0.5 M NaCl, 0.2 M imidazol, pH 7.4

Preparation of the Gel

The gel was prepared according to instructions supplied by themanufacturer.

-   1. The column was washed with 50 mM EDTA, 2 ml-   2. Wash with water, 5 ml-   3. Charging with 0.1 M NiCl₂, 2 ml-   4. Wash with water, 5 ml-   5. Wash with binding buffer, 5 ml

Subsequently the column was opened, the gel recovered and made to 50%slurry by addition of binding buffer.

SDS-PAGE and Staining

SDS-PAGE: method SDS 10-15

CBB staining: method SDS

Silver staining: method Ag

Methods refer to methods in the Phast electrophoresis equipment.

Purification Using the Inventive Apparatus

-   1. 25 μl lysate was dispensed to 7 wells in a PCR-plate (column A)-   2. 25 μl binding buffer was added to each well-   3. 10, 5, 4, 3, 2, 1 and 0.5 μl gelslurry was added to well A-G,    respectively-   4. The plate was incubated for 5 min on a shaker-   5. The inventive apparatus was used to aspirate the gel from the    used wells-   6. The gel was washed for 10-20 s by transfering the inventive    apparatus to through containing binding buffer-   7. Bound proteins were eluted by transfering the inventive apparatus    to a fresh PCR-plate containing 50 μl elution buffer-   8. A sample was withdrawn from each well for analysis. A sample of    the starting material, i.e. lysate, was also taken.-   9. SDS-PAGE was performed followed by staining with CBB or silver    Results

Analysis by SDS-PAGE shows that HisKlenow binds to the gel and can berecovered by treatment with elution buffer. See FIGS. 4 a-b. Both imageshave been manipulated to increase visibility of the scanned image.Numbers indicate the volume (μl) of gel (50% slurry) added to each well.“lys” indicates the starting material.

Discussion

In an initial experiment, a normal 96-well plate with much larger wellswas used for binding and elution. It was obvious from this experimentthat not much gel could aspirated from these much wider wells sinceremaining gel was clearly visible in the wells upon drying.

The subsequent experiment was performed in a PCR plate with concurrentvariation of added gel volume. As can be seen in the FIGS. 4 a-b, therecovery increases with reduction of amount of added gel (see FIG. 4 a,CBB stain). This is probably a reflection of that the subset ofrecovered gel is larger when low amounts of gel is used, leading torecovery of more protein. Thus it seems that the binding capacity of thegel is not a problem in this experiment.

The purification efficiency is very good since no contaminants can befound in the eluted fractions as compared to the starting material (seeFIG. 4 b, Silver stained).

Conclusions

The inventive apparatus can be successfully used for isolation ofproteins from complex mixtures, in this case a bacterial lysate. In thisstudy, a His-tagged protein combined with a metal affinity gel was usedas a model, but the principle should also work with other systems, e.g.an affinity gel, or protein-protein, or receptor ligand interactions.

The applied system was not optimized in any way which is a reason tosuspect that improvements can be made with respect to both recovery andspeed.

1. A method for separating carriers from a solution, said carriers being adapted to have biomolecules, such as DNA, RNA, proteins, polypeptides or carbohydrates attached thereto, wherein the method comprises the steps of: introducing a tubular member (13) into a receptacle which holds a solution containing said carriers; immersing an end portion of the tubular member in the solution; attracting and holding said carriers to said end portion; removing said tubular member together with the held carriers from the solution; characterised in that said attracting and holding step comprises the step of: providing an under-pressure within the tubular member so as to attract and hold the carriers to a filter (25) being disposed in the end portion of the tubular member.
 2. Method according to claim 1, further comprising the step of: releasing said carriers from the filter by removing the under-pressure or creating an over-pressure within the tubular member.
 3. Method according to claim 1, further comprising the step of: releasing said carriers from the filter by means of shaking.
 4. Method according to claim 1, further comprising the step of: releasing said carriers from the filter by means of scraping.
 5. Method according to claim 1, each step being performed simultaneously at a plurality of locations.
 6. Method according to claim 1, wherein said method is used for purifying proteins.
 7. Method according to claim 6, wherein said method is used for purifying tagged proteins.
 8. Method according to claim 7, wherein said method is used for purifying His-tagged proteins.
 9. An apparatus for moving carriers between different receptacles, said carriers being adapted to have biomolecules, such as DNA, RNA, proteins, polypeptides or carbohydrates attached thereto, wherein the apparatus comprises: at least one tubular member (13) to be introduced into a receptacle, which holds a solution containing said carriers, said tubular member having an end portion to be immersed in the solution; means for attracting and holding said carriers to said end portion; characterised in that said means comprises a filter (25) being disposed in the end portion.
 10. Apparatus according to claim 9, further comprising a housing (5) having a sealed inner space (27) which is connectable to a vacuum supply for providing an under-pressure inside the inner space, wherein the tubular member is in fluid communication with said inner space.
 11. Apparatus according to claim 10, wherein the tubular member projects into the inner space.
 12. Apparatus according to claim 9, further comprising a plurality of tubular members having a mutual arrangement corresponding to the arrangement of a multi-well receptacle, such as a Micro Titre Plate (MTP).
 13. Apparatus according to claim 12, wherein all the tubular members are integrated in a separate support unit (23), which is detachable from the housing.
 14. Apparatus according to claim 9, further comprising means for creating an over-pressure within the inner space.
 15. Apparatus according to claim 9, further comprising means for controlling the under-pressure and/or the over-pressure.
 16. Apparatus according to claim 9, wherein the filter is made of sintered material.
 17. Apparatus according to claim 9, wherein the filter is made of sintered polypropylene plastic.
 18. A method for preparing single stranded DNA (ssDNA) from double stranded DNA (dsDNA) comprising the steps of: immobilising one of two strands of said dsDNA on carriers in a first receptacle holding a first solution, wherein the solution contains said carriers and said dsDNA; attracting and holding said carriers to a tubular member (13) being immersed in the first solution; moving said tubular member together with the held carriers from the first receptacle to a second receptacle; separating the dsDNA into ssDNA in the second receptacle; removing the tubular member from the second receptacle, thus removing carriers with immobilised ssDNA from the second receptacle, characterised in that said attracting and holding step comprises the step of : providing an under-pressure within the tubular member so as to attract and hold the carriers to a filter (25) being disposed in the tubular member.
 19. Method according to claim 18, each step being performed simultaneously at a plurality of locations.
 20. Disposable unit adapted to be attached to the tubular member of an apparatus according to the preamble of claim 9, characterised in that said disposable unit is provided with a filter (25) for holding said carriers.
 21. (canceled) 